Optogenetic manipulation of BMP signalling to drive chondrogenic differentiation of hPSCs
Optogenetic manipulation of BMP signalling to drive chondrogenic differentiation of hPSCs
Briefly, adherent cells were dissociated, harvested and centrifuged at 16,000xg. The cell pellet was then resuspended in 300μl lysis buffer. Cell lysate was then transferred to a gDNA removal column and spun at 16,000xg for 30 seconds to remove genomic DNA. The flow-through was collected and RNA was precipitated with the addition of 300μl pure ethanol (≥95%). Total RNA was then transferred to an RNA purification column and subjected to a series of on-column washes and centrifugations before elution in nuclease-free water (Invitrogen, #4387937) according to the manufacturer's instructions. This submission contains human embryonic stem cells (hESCs) and differentiated samples. An optogenetic bone morphogenetic protein (BMP) signalling system (optoBMP) was used to drive chondrogenic differentiation of human embryonic stem cells (hESCs). The human embryonic stem cell line MAN13 is a clinical grade hESC line which has been well characterised (PMID: 28583200) (https://hpscreg.eu/cell-line/UMANe002-A) and was derived under local ethical approval (Central Manchester LREC favourable opinion 03/CM/684) and UK Human Fertilisation and Embryology Authority license (HFEA licence R0171) with fully informed parental consent. Here MAN13 was modified with an optogenetic BMP signalling system that functions through light-induced dimerisation of light-oxygen-voltage (LOV) domains fused to the intracellular regions of BMPR1B/ALK6 and BMPR2 (Humphreys, P. A., Woods, S., Smith, C. A., Bates, N., Cain, S. A., Lucas, R. & Kimber, S. J. Optogenetic control of the BMP signaling pathway. ACS Synthetic Biology 9, 3067–3078 (2020)). Samples for RNA-seq analysis of optoBMP-hESC-derived chondroprogenitors were collected at day 11 after differentiation with either light (LIGHT), a combination of BMP2 and GDF5 (B2/G5) or after being kept in dark conditions without additional BMP pathway activation (DARK). OptoMAN13 were subjected to chondrogenic differentiation as previously described (Mancini, F. E., Humphreys, P. E. A., Woods, S., Bates, N., Cuvertino, S., O'Flaherty, J., Biant, L., Domingos, M. A. N. & Kimber, S. J. Effect of a retinoic acid analogue on BMP-driven pluripotent stem cell chondrogenesis. bioRxiv (2023). at <https://doi.org/10.1101/2023.06.20.545738>). OptoMAN13 cells were seeded in 24 well plates at 5x104 cells/well and maintained in dark conditions prior to optogenetic stimulation. Optical stimulation was performed using a custom-built Arduino-controlled LED array device using blue 488nm LEDs within a standard cell culture incubator at 37°C (5% Co2, 20% O2). Optogenetic stimulation was achieved by intermittent illumination of optoMAN13 cells (1 hour illumination, 3 hours dark) at 1.5mW/cm2 irradiance. Total RNA was extracted using Monarch® Total RNA Miniprep Kit (New England Biolabs inc. #T2010S) following manufacturer protocol.  Stranded mRNA protocol